The present invention generally relates to protocols for evaluating the hemostasis of a patient at risk of developing thrombosis, and specifically, to diagnostic assays for determining the level of antithrombin III present in a plasma sample withdrawn from a patient. The invention particularly relates, in a preferred embodiment, to a chromogenic thrombin-based (ie, type IIa) assay for determining antithrombin III. The invention also relates to a heparin derivative, to a reagent composition and to a high-calibrator plasma reference useful in such assays and to kits employing the same.
The advantageous formation of blood clots during wound healing and the undesirable development of thrombi in connection with thrombosis both involve the proteolytic action of the serine protease thrombin on fibrinogen. The level of thrombin present in vivo is primarily regulated by the heparin-catalyzed thrombin inhibitor, antithrombin III (ATIII). Hence, the level of ATIII present in vivo is of significant clinical importance for diagnosing and monitoring patients at risk for excessive bleeding, due to abnormally high levels of ATIII, or at risk for developing thrombi, due to abnormally low levels of ATIII.
One type of assay for antithrombin III is based on the capability of ATIII present in a plasma sample to inhibit the proteolytic activity of exogenously added thrombin in the presence of heparin. The residual, uninhibited thrombin is then determined by methods typically involving, for example, thrombin-specific chromogenic substrates and spectrophotometric analyses, or alternatively, fibrinogen and clot-forming analyses.
However, such thrombin-based ATIII assays--also referred to as factor IIa ATIII assays--are inaccurate due to the activity of a second heparin-catalyzed thrombin-inhibitor, heparin cofactor II (HCII). See Tollefson et al., Heparin Cofactor II, Jrnl. Biol. Chem. 257:5, pp. 2162-2169 (1982). That is, thrombin-based assays known in the art measure the levels of exogenous thrombin remaining after the thrombin has reacted with both ATIII and HCII--not just with ATIII. The inaccuracy of such assays are of particular clinical concern for ATIII-deficient patients. In cases where HCII is in the high normal range and ATIII is determined using thrombin-based assays known in the art, an ATIII deficiency can be masked by the HCII antithrombin activity.
Several approaches have been developed for avoiding the masking effect of HCII. One proposed approach for a factor IIa assay involved the use of bovine thrombin in the presence of relatively low concentrations of heparin. See Friberger et al., Antithrombin Assay--The Use of Human or Bovine Thrombin and the Observation of a "Second" Heparin Cofactor, Thrombosis Research 25, pp. 433-436 (1982). This approach, however, did not provide a commercially recognized solution to the problem.
Another approach is described in U.S. Pat. No. 5,646,007 to Enomoto et al., wherein a chromogenic thrombin-based ATIII assay is conducted in the presence of salt at a concentration ranging from about 0.2 M to about 0.9 M, and preferably at a concentration greater than about 0.3. While this approach offers some benefits with respect to limiting the effect of HCII activity against thrombin, the sensitivity of chromogenic assays for thrombin decreases as the concentration of salt increases, due to an allosteric effect on thrombin in the presence of such higher salt concentrations. Reduced sensitivity is of particular commercial significance for ATIII assays performed using automated analyzers.
According to yet another approach, the ATIII assay is performed using exogenously added factor Xa as an indicator instead of thrombin. See Demers et al., An Antithrombin III Assay Based on Factor Xa Inhibition Provides A More Reliable Test to Identify Congential Antithrombin III Deficiency Than an Assay Based on Thrombin Inhibition, Thrombosis and Hemostasis 69:3 pp. 231-235 (1993). Unlike thrombin, factor Xa is not inhibited by HCII. However, factor Xa-type ATIII assays are more expensive than factor IIa-type assays. Moreover, factor Xa can be less stable than thrombin under certain conditions, and the factor Xa-based assays can require a greater degree of dilution. As such, factor Xa ATIII assays are less suitable for use with automated analyzers.
Assays for determining ATIII, and in fact, assays for determining other plasma constituents, have other common problems when the assay protocol involves correlation of a determined amount using a standard curve developed from reference plasma samples of known constituent concentration. Because reference plasmas known in the art typically have high-end constituent concentrations of less than about 105% of normal, it is not possible to directly determine a plasma constituent which is substantially above the normal concentration range of that constituent (e.g. about 110% or more). Instead of making such determination directly, it is presently necessary to dilute the patient plasma sample (e.g. 1:1 in saline), reperform the determination, and then multiply the determined amount by the dilution factor (e.g. 2 for a 1:1 dilution).